Mitochondrial dysfunction may be responsible for the pathogenesis and progression of non-alcoholic fatty liver disease (NAFLD), a condition that affects over 71 million individuals in the U.S. alone. Impaired mitochondrial function could lead to both the initial accumulation of hepatic triglycerides via decreased oxidative disposal, as well as the transition to steatohepatitis due to enhanced oxidative stress originating from the plethora of redox reactions required for oxidative phosphorylation. As a result, we believe that there may be significant differences in mitochondrial metabolism between individuals with bland steatosis, presumed to be a benign condition, and steatohepatitis (NASH), a progressive and morbid form of NAFLD. The goal of this grant will be to use newly-developed, non-invasive, in vivo NMR-based stable isotope methodologies to define the role of hepatic mitochondrial dysfunction in the development of NAFLD and how these alterations relate to the development of insulin resistance and NASH. Such measurements are difficult or impossible to do using standard mass spectrometry techniques or classic radiotracers. The first aim of this proposal seeks to determine if the increased rates of gluconeogenesis observed in insulin resistance and NAFLD are associated with increased energy generation via the citric acid (TCA) cycle, a component of mitochondrial function. We have observed an association between energy generation in the TCA cycle and gluconeogenesis occurring from precursors such as lactate and alanine, suggesting that these processes are energetically linked. In the second aim of this proposal we will determine if progression from bland steatosis to NASH is associated with a progressive decline in a second component of mitochondrial function, ketogenesis. Several reports demonstrate that FA synthesis in liver is inappropriately increased in subjects with NAFLD, implying that mitochondrial FA 2-oxidation is inhibited via the effect of malonyl-CoA on carnitine palmitoyl transferase. Attenuation of hepatic 2-oxidation may be associated with decreased export of ketones to the periphery in favor of local energy generation to support cellular processes in the hepatocyte. In the final aim, the ability of metformin, a proposed therapy for NAFLD, to augment hepatic mitochondrial function in individuals with NAFLD will be quantified. The benefits of the proposed research are several-fold: First, improved understanding of the metabolic derangements complicit in the pathogenesis and progression of NAFLD will allow better clinical insight and focused approaches to therapy; Second, our methodologies may provide a simple, non-invasive method to identify individuals with NASH, who are at greatest risk of progressive liver disease; and Third, a better understanding of the mechanism of action of metformin in the treatment of NAFLD will allow this therapy to be targeted to individuals most likely to derive benefit. PUBLIC HEALTH RELEVANCE: Accumulation of fat in the liver due to obesity and diabetes is termed non-alcoholic fatty liver disease (NAFLD), with over 71 million individuals in the USA afflicted with this disorder. Such fat accumulation can lead to inflammation and progressive liver disease in some individuals. The goal of this proposal is to determine if impaired mitochondrial function contributes to the pathogenesis and progression of NAFLD using non-invasive techniques based on stable isotope tracers and nuclear magnetic resonance spectroscopy, techniques that have the potential to immediately impact clinical care by providing a method to identify individuals at risk for progressive liver disease, heretofore only possible by liver biopsy.